WO2015005307A1

WO2015005307A1 - Method for producing saccharide solution

Info

Publication number: WO2015005307A1
Application number: PCT/JP2014/068115
Authority: WO
Inventors: 栗原　宏征; 裕子石塚; 山田　勝成
Original assignee: 東レ株式会社
Priority date: 2013-07-09
Filing date: 2014-07-08
Publication date: 2015-01-15
Also published as: AU2014288309B9; BR112015032515A2; CN105452478A; MY180037A; EP3020820A4; US9976160B2; JPWO2015005307A1; CA2917307C; JP6459514B2; CN110499396A; US10815501B2; EP3020820A1; US20160145651A1; AU2014288309B2; AU2014288309A1; CA2917307A1; US20180237805A1; BR112015032515A8; CN105452478B

Abstract

[Problem] An aqueous saccharide solution obtained from biomass contains various fermentation-inhibiting substances, however, it is possible to easily remove fermentation-inhibiting substances derived from biomass at a low cost by treating the aqueous saccharide solution with microbes that do not utilize glucose or xylose or with a crude enzyme product derived from said microbes.

Description

Method for producing sugar solution

The present invention relates to a method for producing a sugar liquid from biomass.

The fermentation production process of chemicals using sugar as a raw material is used for the production of various industrial raw materials. Currently, sugar derived from edible raw materials such as sugar cane, starch and sugar beet is used industrially as sugar for this fermentation raw material, but the price of edible raw materials will rise due to the increase in the world population in the future, or it will compete with edible foods. From the ethical side of the process, a process for producing sugar solution more efficiently than renewable non-edible resources, ie cellulose-containing biomass, or a process for efficiently converting the obtained sugar solution as a fermentation raw material into an industrial raw material The future is a future challenge.

As a method for producing a sugar solution from cellulose-containing biomass, a method of producing a sugar solution by acid hydrolysis of cellulose and hemicellulose using concentrated sulfuric acid (Patent Document 1 or 2), hydrolysis of cellulose-containing biomass with dilute sulfuric acid A method for producing a sugar solution by further treatment with an enzyme such as cellulase after the treatment is disclosed (Non-patent Document 1). In addition, as a method that does not use an acid, a method of hydrolyzing cellulose-containing biomass by using subcritical water at about 250 to 500 ° C. to produce a sugar solution (Patent Document 3), or treating cellulose-containing biomass with subcritical water After that, a method for producing a sugar solution by further enzyme treatment (Patent Document 4), and a cellulose-containing biomass is hydrolyzed with pressurized hot water at 240 to 280 ° C., followed by further enzyme treatment. A method for producing a liquid (Patent Document 5) is disclosed.

However, in hydrolysis of biomass containing cellulose, the decomposition reaction of sugars such as glucose and xylose proceeds simultaneously with the decomposition of cellulose or hemicellulose components, furan compounds such as furfural and hydroxymethylfurfural, formic acid, acetic acid and levulin. There was a problem of generating by-products such as organic acids such as acids. In addition, since the cellulose-containing biomass contains a lignin component that is an aromatic polymer, the lignin component is decomposed in the acid treatment step, and an aromatic compound such as a low molecular weight phenol is simultaneously generated as a byproduct. These compounds act as inhibitors in the fermentation process using microorganisms, cause microbial growth inhibition, and reduce the yield of fermentation products. It was a big problem when using as.

As a method for removing such fermentation-inhibiting substances during the sugar liquid production process, a method such as overlining has been disclosed (Non-Patent Document 2). In this method, in the step of adding and neutralizing lime to cellulose or saccharified solution after acid treatment, a fermentation inhibitor such as furfural and HMF is retained in the gypsum component while being heated to around 60 ° C. for a certain period of time. It is a method of removing together. However, the overlining has a problem that the effect of removing organic acids such as formic acid, acetic acid and levulinic acid is small.

Further, as another method for removing the fermentation inhibitory substance, a method of evaporating and removing the fermentation inhibitory substance by blowing water vapor into the sugar solution from the cellulose-containing biomass is disclosed (Patent Document 6). However, such evaporation and removal methods depend on the boiling point of the fermentation inhibitor, and in particular, the removal efficiency of fermentation inhibitors such as organic acids having a low boiling point is low. To obtain sufficient removal efficiency, a large amount of energy is required. There was a problem that it had to be thrown in.

In addition, there is a method for removing fermentation-inhibiting substances by ion exchange (Patent Document 7), but there is a problem in terms of cost, and there is also a method for adsorption removal using a wood-based carbide, that is, activated carbon. There was a problem that it was limited to a hydrophobic compound (Patent Document 8).

In addition, although a method for removing a fermentation inhibitor as a permeate through a membrane using a nanofiltration membrane or a reverse osmosis membrane has been devised, there is a problem that additional separation equipment and energy input are necessary for the removal. (Patent Document 9).

Japanese National Patent Publication No. 11-506934 JP 2005-229821 A Japanese Patent Laid-Open No. 2003-212888 JP 2001-95597 A Japanese Patent No. 3041380 JP 2004-187650 A JP-T-2001-511418 JP 2005-270056 A Japanese Patent No. 4770987

As described above, the fermentation inhibitor contained in the sugar solution derived from biomass inhibits the growth and metabolic conversion of microorganisms. Therefore, in order to remove these fermentation inhibitors, adsorption treatment, ion exchange, heat evaporation, nanofiltration membrane However, there has been a problem that the processing cost is high or the removable fermentation inhibitor is limited to a specific compound.

That is, the problem to be solved by the present invention is to provide a method for producing a sugar solution including a step of comprehensively removing various fermentation-inhibiting substances at a low cost with a simple treatment.

The present inventor is able to reduce the concentration of a fermentation inhibitor contained in a biomass-derived sugar solution by utilizing the metabolic mechanism of a specific microorganism, and that the obtained sugar solution can be used as a fermentation raw material. The headline and the present invention were completed.

That is, the present invention includes the following [1] to [15].
[1] A method for producing a sugar solution from biomass, which is a fermentation inhibitor contained in an aqueous sugar solution obtained from biomass, such as coumaric acid, coumaric acid, ferulic acid, ferulamide, vanillin, vanillic acid, acetovanillone, furfural and 3 -A step of decomposing one or more compounds selected from the group consisting of hydroxymethylfurfural with a microorganism that does not assimilate glucose and / or xylose or a crude enzyme derived from the microorganism; Production method.
[2] The method for producing a sugar liquid according to [1], wherein the microorganism that does not assimilate glucose and / or xylose is a Delftia sp.
[3] The glucose and / or xylose non-assimilating microorganism may be selected from Delftia acidovorans, Delftia lacustris, Delftia tsuruhatensis and Delphia d. The method for producing a sugar liquid according to [1] or [2], which is one or more selected from the group consisting of:
[4] The method for producing a sugar solution according to any one of [1] to [3], wherein the sugar solution is a sugar solution obtained by hydrolyzing cellulose-containing biomass.
[5] The method for producing a sugar solution according to [4], including a step of preparing an aqueous sugar solution by one or more treatments selected from the group consisting of acid treatment, alkali treatment, hydrothermal treatment and enzyme treatment for cellulose-containing biomass.
[6] The method for producing a sugar liquid according to any one of [1] to [3], wherein the sugar aqueous solution is molasses.
[7] The method for producing a sugar liquid according to any one of [1] to [6], wherein the fermentation inhibiting substance decomposition treatment step is a treatment at a monosaccharide concentration of less than 100 g / L.
[8] The method for producing a sugar solution according to any one of [1] to [7], wherein the fermentation inhibiting substance decomposition treatment step is a treatment within a pH range of 6 to 11.
[9] A method for producing a sugar solution, wherein the sugar concentration obtained by the method for producing a sugar solution according to any one of [1] to [8] is increased by membrane concentration and / or evaporation concentration.
[10] A method for producing a sugar solid, wherein a sugar solid obtained by the method for producing a sugar solution according to any one of [1] to [8] is obtained by membrane concentration and / or evaporation concentration.
[11] A sugar solution obtained by the method for producing a sugar solution according to any one of [1] to [9].
[12] A saccharide solid obtained by the method for producing a saccharide solid according to [10].
[13] A sugar solution or sugar solid derived from cellulose-containing biomass or molasses, which is selected from the group consisting of impurities such as serine, threonine, asparagine, aspartic acid, glutamine, glutamic acid, proline, phenylalanine, lysine and histidine A sugar solution or a sugar solid, wherein the content of the seed or two or more free amino acids is below the detection limit.
[14] A step of obtaining a sugar solution by the method for producing a sugar solution according to any one of [1] to [9], and culturing microorganisms using the obtained sugar solution as a fermentation raw material to convert the sugar into a chemical product A method for producing a chemical product, comprising a step of converting.
[15] A chemistry process comprising: obtaining a saccharide solid by the method for producing a saccharide solid according to [10]; and culturing a microorganism using the obtained saccharide solid as a fermentation raw material to convert the saccharide into a chemical product. Product manufacturing method.

The sugar solution obtained in the present invention can be used as a fermentation raw material for microorganisms and can be used as a raw material for various chemical products.

FIG. 1 is a flowchart showing the procedure for carrying out the method for producing a sugar liquid of the present invention. FIG. 2 is a flow chart showing a preferred implementation procedure of the method for producing a sugar liquid of the present invention. FIG. 3 is a drawing that traces the time course of ferulamide by Delftia thulhatensis. FIG. 4 is a drawing that traces the time course of ferulamide by Delphthia acidborans. FIG. 5 is a drawing that traces the time course of coumarinamide due to Delftia thurhatensis. FIG. 6 is a drawing that traces the change with time of Kumaramide by Delphthia acidborans. FIG. 7 is a photograph showing the change over time of the sugar solid of the present invention.

The present invention is a method for producing a sugar liquid from biomass. Biomass is classified into sugar-producing crops and grains containing sugar or polysaccharides and cellulose-containing biomass. Examples of sugar-producing crops / cereals include sugarcane, sweet potato, corn, sugar beet, cassava, rice, wheat, soybean, and the like. Cellulose-containing biomass includes bagasse, corn stover, corn cob, trees, bark, wood chips, waste building materials, EFB (Empty fruits bunch), eggplant gala, elephant grass, napier grass, Eliansus, switch grass, wheat straw, rice straw, Examples include warm bamboo, bamboo, and coffee / tea bowls. That is, the sugar aqueous solution in the present invention is a sugar solution using such biomass as a raw material, and is a sugar aqueous solution obtained from the aforementioned biomass through steps such as extraction, concentration, hydrolysis, and crystallization, and at least described later. This refers to an aqueous sugar solution containing the sugar and fermentation inhibitor.

Such sugar aqueous solutions include molasses (molasses: mother liquor after sugar crystallization or its concentrate) obtained in the sugar production process from sugar-producing crops and grains, and sugar aqueous solutions obtained by hydrolyzing cellulose-containing biomass. A preferred example is given.

The sugar aqueous solution contains a fermentation inhibitor as an impurity. Fermentation inhibitors are compounds derived from the components contained in the aforementioned biomass, or produced by chemical conversion from the aforementioned biomass, and are compounds that cause an inhibitory effect on fermentation production by microorganisms. Refers to that. The term “inhibitory” as used herein means that, due to the presence of a fermentation inhibitor, 1) sugar consumption by microorganisms is delayed, or 2) growth of microorganisms is delayed, and 3) the production of fermentation products of microorganisms is reduced. It means to do. With regard to these 1) to 3), the presence or absence of inhibition can be confirmed by comparing on the basis of a medium containing no fermentation inhibitor. Further, specifically, the fermentation inhibitor may be exemplified by furan compounds, aromatic compounds, organic acids, etc. In particular, in the present invention, it is reduced by decomposing furan compounds and aromatic compounds. be able to. A furan compound refers to a group of compounds having a furan skeleton, and examples thereof include furfural and hydroxymethylfurfural. The former is produced by converting xylose from the latter into glucose. As an aromatic compound, it is a compound which has an aromatic ring, Comprising: Vanillin, vanillic acid, syringic acid, coumaric acid, coumaramide, ferulic acid, ferulamide, etc. can be illustrated.

The aqueous sugar solution used in the present invention is one or two selected from the group consisting of coumaric acid, coumaric amide, ferulic acid, ferulamide, vanillin, vanillic acid, acetovanillone, furfural and 3-hydroxymethylfurfural as fermentation inhibitors. An aqueous sugar solution containing the above compound. Whether these fermentation-inhibiting substances are contained in the aqueous sugar solution corresponds to the sugar solution by separating the above-mentioned preparation of fermentation-inhibiting substances by reversed-phase chromatography (HPLC) and holding time under specific separation conditions. It can be determined whether a fermentation inhibitor is included. In reverse phase chromatography, a chromatograph can be obtained by measuring the absorbance of the eluate at 180 to 400 nm for each retention time. A calibration curve is prepared in advance using a preparation of fermentation inhibitor, and the concentration of the fermentation inhibitor contained in the sugar solution is determined based on the peak area or peak height in the sugar solution. be able to. Such analysis may be performed according to the analysis method or apparatus to be used by concentrating or diluting the sugar solution.

The aqueous sugar solution contains one or more fermentation inhibitors selected from the group consisting of coumaric acid, coumaric amide, ferulic acid, ferulamide, vanillin, vanillic acid, acetovanillone, furfural and 3-hydroxymethylfurfural. Means that these substances are contained in the sugar aqueous solution within a detectable range, and preferably 1 mg / mL or more, more preferably 10 mg / mL or more, and further preferably 100 mg / L or more. Moreover, in the manufacturing method of the sugar liquid in this invention, two or more of these fermentation inhibitory substances may be contained, and 2 or more types, 3 or more types, and 4 or more types may be contained.

The sugar aqueous solution contains at least sugar. A sugar refers to a component that dissolves in water among monosaccharides and polysaccharides, and includes at least a monosaccharide consisting of one such sugar, a disaccharide, or a trisaccharide. Specific examples of the sugar include glucose, xylose, arabinose, xylitol, arabitol, sucrose, fructose, lactose, galactose, mannose, cellobiose, cellotriose, xylobiose, xylotriose, maltose, trehalose, and the like. The sugar concentration in the aqueous sugar solution of the present invention is 0.01 g / L or more, preferably 0.1 g / L or more, more preferably 1 g / L or more, and most preferably 10 g / L or more. Is included.

Furthermore, the aqueous sugar solution contains inorganic salts, organic acids, amino acids, vitamins, etc., in addition to the aforementioned fermentation inhibitor and sugar. Examples of inorganic salts include potassium salts, calcium salts, sodium salts, magnesium salts, and the like. Examples of the organic acid include lactic acid, citric acid, acetic acid, formic acid, malic acid, succinic acid and the like. Examples of amino acids include glutamine, glutamic acid, asparagine, aspartic acid, glycine, alanine, phenylalanine, tyrosine, tryptophan, arginine, methionine, cysteine, histidine, leucine, isoleucine, lysine, proline, serine, threonine, valine, etc. it can.

When the biomass is cellulose-containing biomass, the aqueous sugar solution can be obtained by hydrolyzing the cellulose-containing biomass in one or more treatments selected from the group consisting of acid treatment, alkali treatment, hydrothermal treatment, and enzyme treatment. Hydrolysis is a treatment that hydrolyzes polysaccharides such as cellulose and hemicellulose contained in cellulose-containing biomass into monosaccharides or oligosaccharides, and the hydrolyzate contains fermentation inhibitors in addition to these monosaccharides or oligosaccharides. It is.

Acid treatment is performed by adding an acid such as sulfuric acid, acetic acid, hydrochloric acid, or phosphoric acid to the biomass. In the acid treatment, hydrothermal treatment may be performed. In the case of the sulfuric acid treatment, the concentration of sulfuric acid is preferably 0.1 to 15% by weight, and more preferably 0.5 to 5% by weight. The reaction temperature can be set in the range of 100 to 300 ° C., preferably 120 to 250 ° C. The reaction time can be set in the range of 1 second to 60 minutes. The number of processes is not particularly limited, and the process may be performed once or more. In particular, when the above process is performed twice or more, the first process and the second and subsequent processes may be performed under different conditions.

Alkali treatment is performed by adding alkali such as ammonia, caustic soda, potassium hydroxide to the biomass. In the alkali treatment, hydrothermal treatment may be performed. The ammonia treatment is in accordance with the method described in JP2008-161125A or JP2008-535664A. For example, the ammonia concentration to be used is added in the range of 0.1 to 15% by weight with respect to the biomass, and the treatment is performed at 4 to 200 ° C., preferably 90 to 150 ° C. Ammonia to be added may be in a liquid state or a gaseous state. Further, the form of addition may be pure ammonia or an aqueous ammonia solution. The number of processes is not particularly limited, and the process may be performed once or more. In particular, when the process is performed twice or more, the first process and the second and subsequent processes may be performed under different conditions.

Hydrothermal treatment is a method of treating cellulose-containing biomass by adding only water and heating without adding acid and alkali. In the case of hydrothermal treatment, after adding water so that the biomass becomes 0.1 to 50% by weight, it is treated at a temperature of 100 to 400 ° C. for 1 second to 60 minutes. By treating at such temperature conditions, hydrolysis of cellulose occurs. The number of processes is not particularly limited, and the process may be performed once or more. In particular, when the process is performed twice or more, the first process and the second and subsequent processes may be performed under different conditions.

Enzyme treatment is a hydrolysis treatment by adding saccharifying enzyme to cellulose-containing biomass. The pH for hydrolysis with a saccharifying enzyme is preferably in the range of pH 3 to 9, more preferably pH 4 to 5.5, and even more preferably pH 5. For pH adjustment, acid or alkali can be added and adjusted so as to have a desired pH. Moreover, you may use a buffer solution suitably. In the enzyme treatment, it is preferable to carry out stirring and mixing in order to promote contact between cellulose and the enzyme and to make the sugar concentration of the hydrolyzate uniform. The cellulose is preferably added so that the solid content concentration is in the range of 1 to 25% by weight, and more preferably in the range of 5 to 20% by weight.

As the saccharifying enzyme, a filamentous fungus-derived cellulase can be preferably used. Examples of the filamentous fungi include Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Humicora, and Humicola. Ilpex (Irpex), Mucor, Mucor, Talaromyces, Phanerochaete, white decay gold, brown decay fungus, and the like can be exemplified. Among these filamentous fungus-derived cellulases, it is preferable to use a Trichoderma-derived cellulase having a high cellulose-degrading activity.

Trichoderma-derived cellulase is an enzyme composition mainly composed of cellulase derived from Trichoderma microorganisms. The microorganism of the genus Trichoderma is not particularly limited, but Trichoderma reesei (Trichoderma reesei) is preferable, and specifically, Trichoderma reesei QM9414 (Trichoderma reesei QM9414), Trichoderma reesei QM9123 (Trichoderma reesei QM9123) reeseiRut C-30), Trichoderma reesei PC3-7 (Trichoderma reesei PC3-7), Trichoderma reesei CL-847 (Trichoderma reeseiCL-847), Trichoderma reesei MCG77 (Trichoderma MCe 77) Ma reesei MCG80 (Trichoderma reeseiMCG80), can be exemplified Trichoderma viride QM9123 a (Trichoderma viride9123). Further, it may be a microorganism derived from the aforementioned genus Trichoderma, which has been subjected to a mutation treatment with a mutation agent or ultraviolet irradiation to improve cellulase productivity.

Trichoderma-derived cellulase is an enzyme composition containing a plurality of enzyme components such as cellobiohydrase, endoglucanase, exoglucanase, β-glucosidase, xylanase, and xylosidase and having an activity of hydrolyzing and saccharifying cellulose. Trichoderma-derived cellulase can efficiently hydrolyze cellulose due to the concerted effect or complementary effect of a plurality of enzyme components in cellulose degradation. In particular, the cellulase used in the present invention preferably contains Trichoderma-derived cellobiohydrase and xylanase.

Cellobiohydrase is a general term for cellulases characterized by hydrolysis from the terminal portion of cellulose. The enzyme group belonging to cellobiohydrase is represented by EC number: EC3.2.1.91. Are listed.

Endoglucanase is a general term for cellulases characterized by hydrolysis from the central part of the cellulose molecular chain. EC numbers: EC 3.2.1.4, EC 3.2.1.6, EC 3.2.1 .39, EC 3.2.1.73, an enzyme group belonging to endoglucanase is described.

Exoglucanase is a general term for cellulases characterized by hydrolysis from the end of a cellulose molecular chain, and is assigned to the exoglucanase as EC numbers: EC3.2.1.74 and EC3.2.1.58. Enzyme groups are described.

Β-glucosidase is a general term for cellulases characterized by acting on cellooligosaccharide or cellobiose, and an enzyme group belonging to β-glucosidase is described as EC number: EC 3.2.1.21.

Xylanase is a general term for cellulases characterized by acting on hemicellulose or particularly xylan, and an enzyme group belonging to xylanase is described as EC number: EC3.2.1.8.

Xylosidase is a general term for cellulases characterized by acting on xylo-oligosaccharides, and an enzyme group belonging to xylosidase is described as EC number: EC 3.2.1.37.

As the Trichoderma-derived cellulase, a crude enzyme product is preferably used. The crude enzyme product is derived from a culture supernatant obtained by culturing the microorganism for an arbitrary period in a medium adjusted so that the microorganism of the genus Trichoderma produces cellulase. The medium components to be used are not particularly limited, but in order to promote the production of cellulase, a medium to which cellulose is added can be generally used. As the crude enzyme product, the culture supernatant is preferably used as it is, or the culture supernatant from which Trichoderma cells are removed.

The weight ratio of each enzyme component in the crude enzyme product is not particularly limited. For example, the culture solution derived from Trichoderma reesei contains 50 to 95% by weight of cellobiohydrase, and the rest. The endoglucanase, β-glucosidase and the like are included in the components. Trichoderma microorganisms produce strong cellulase components in the culture solution, while β-glucosidase is retained in the cell or on the cell surface and therefore has low β-glucosidase activity in the culture solution. A heterogeneous or homologous β-glucosidase may be further added to the enzyme product. As the heterogeneous β-glucosidase, β-glucosidase derived from Aspergillus can be preferably used. Examples of β-glucosidase derived from the genus Aspergillus include Novozyme 188 commercially available from Novozyme. As a method of adding a heterologous or homologous β-glucosidase to a crude enzyme product, a gene is introduced into a Trichoderma microorganism, and the Trichoderma microorganism that has been genetically modified so as to be produced in the culture solution is cultured. A method of isolating the culture solution may also be used.

It is preferable to appropriately combine a plurality of the acid treatment, alkali treatment, hydrothermal treatment, and enzyme treatment described above, and it is more preferred to further carry out the enzyme treatment on the cellulose-containing biomass that has been subjected to the acid treatment, alkali treatment, or hydrothermal treatment.

In the present invention, the fermentation inhibitor contained in the aforementioned aqueous sugar solution is decomposed by a microorganism that does not assimilate glucose and / or xylose, preferably a microorganism that does not assimilate glucose and xylose. The decomposition treatment of the fermentation inhibitory substance means that the fermentation inhibitory substance is accompanied by a chemical structural change due to the action of microorganisms or enzymes, and is that microbial toxicity is reduced by reducing the molecular weight of the fermentation inhibitory substance or by hydroxylation. In the present invention, a microorganism that does not assimilate glucose and / or xylose or a crude enzyme product derived from the microorganism is used in the decomposition treatment of the fermentation inhibitor. Thereby, a wide range of fermentation inhibiting substances can be efficiently decomposed and removed.

The microorganism having no assimilation of glucose and / or xylose in the present invention means xylose and / or glucose under the medium and culture conditions (optimum pH, optimum temperature, optimum aeration condition) in which the microorganism is grown. Is a microorganism characterized in that it is not substantially consumed as a carbon source. Also, for example, Pseudomonas aeruginosa known as an obligate aerobic bacterium assimilate glucose and / or xylose under anaerobic conditions and consume under an aerobic condition. Microorganisms that do not assimilate xylose are also included in the “microorganisms that do not assimilate glucose and / or xylose” in the present invention. It may be a microorganism in which the assimilation of glucose and / or xylose has been eliminated by introducing genetic recombination or gene mutation.

Examples of microorganisms having no assimilation of glucose and / or xylose that can be preferably used in the present invention include Delftia sp., Commamonas sp., Delxomyces sp., Ferromyces sp. Examples include microorganisms belonging to sp), among which microorganisms belonging to the genus Delftia having an excellent ability to decompose fermentation inhibitors are more preferable.

Examples of microorganisms belonging to the genus Delftia include Delftia acidvorans, Delftia lacustris, Delftia tsuruhatensis, and Delftia litupae.

Whether or not the microorganism is a Delphtia microorganism is determined by determining the 16S rDNA base sequence of the microorganism to be identified, and the 16S rDNA base sequence of Delftia lacustris 322 (Accession No. EU888308). A sequence comparison is performed, and if the sequence identity is 93% or more, it can be identified as Delphthia.

Although there are microorganisms that are not attributed to the genus Delphthia and have different genus names as microorganisms identified by the above-described identification method, they have characteristics that do not assimilate glucose and / or xylose, and that decompose degradation of fermentation inhibitors. As long as it has, it can be used for the manufacturing method of the sugar liquid of this invention as what is contained in the Delphia microorganisms in this invention. Specific microbial genus names including microorganisms having the property that the sequence of 16S RNA may be contained as 93% or more of microorganisms and have the property of degrading fermentation inhibitory substances include Comamonas and Acid Borak. Acidovorax, Giesbergeria, Simpliciera, Alycicliphilus, Diaphorobacter, Tepidocera (Tepideocella) A genus (Brachymonas) etc. can be illustrated. These are all microorganisms closely related to the genus Delphthia and may be used in the present invention.

In addition, the crude enzyme product derived from a microorganism that does not assimilate glucose and / or xylose refers to an enzyme component derived from the microorganism and a component containing two or more enzyme components. Such a crude enzyme product can be prepared by culturing a microorganism having no assimilation of glucose and xylose in an appropriate medium and extracting the crude enzyme solution from the cultured cells. Alternatively, a gene of a microorganism that does not assimilate glucose and / or xylose can be isolated, introduced into a suitable host, and expressed as a heterologous recombinant protein. Preferably, a microorganism that does not assimilate glucose and / or xylose is cultured, and an enzyme is extracted from this microorganism. It is preferable that no special purification operation is performed. Since the crude enzyme product contains two or more kinds of enzyme components, a plurality of fermentation-inhibiting substances can be decomposed simultaneously into compounds with reduced inhibition.

In the decomposition treatment of the fermentation inhibitor, the aqueous sugar solution containing the fermentation inhibitor described above and the microorganism or the crude enzyme product derived from the microorganism are mixed and incubated at the optimum growth temperature or optimum growth pH of the microorganism. Is done. As specific temperature conditions, a range of 20 to 40 ° C. is preferable, and a range of 25 to 32 ° C. is more preferable. The pH condition is preferably in the range of pH 6.5 to 10, and more preferably in the range of pH 7 to 8.5.

The fermentation inhibitor to be decomposed is at least one selected from the group consisting of coumaric acid, coumarinamide, ferulic acid, ferulamide, vanillin, vanillic acid, acetovanillone, furfural, 3-hydroxymethylfurfural, preferably two or more, More preferably, they are 3 or more types, More preferably, they are 4 or more types.

In the present invention, solids contained in the sugar solution may be removed in advance by solid-liquid separation. The method of solid-liquid separation is not particularly limited, but membrane separation by centrifugation such as a screw decanter, filter press or microfiltration membrane (microfiltration) is preferred, and membrane separation is more preferred.

Further, the aqueous sugar solution may be subjected to an ultrafiltration membrane (UF membrane) treatment. Although the method of ultrafiltration membrane treatment is not particularly limited, the ultrafiltration membrane to be used should be an ultrafiltration membrane having a molecular weight cut-off of 500 to 200,000 Da, preferably 10,000 to 50,000 Da. Can do. In particular, when the sugar solution used contains the enzyme used in hydrolysis of cellulose-containing biomass, it was used for hydrolysis by using an ultrafiltration membrane having a small fractional molecular weight relative to the molecular weight of the enzyme. The enzyme can be separated and recovered.

As a material for the ultrafiltration membrane, a membrane made of a material such as polyethersulfone (PES), polyvinylidene fluoride (PVDF), or regenerated cellulose can be used. As the ultrafiltration membrane shape, a tubular type, a spiral element, a flat membrane or the like can be preferably used. The filtration of the ultrafiltration membrane includes a cross flow method and a dead end filtration method, but the cross flow filtration method is preferable in terms of membrane fouling and flux.

A microorganism having no assimilation of glucose and / or xylose or a crude enzyme product derived from the microorganism may be separated and recovered from the sugar solution after the decomposition treatment of the fermentation inhibitor and reused. The separation and recovery can be performed by appropriately selecting and combining a centrifugal separation, a microfiltration membrane, an ultrafiltration membrane or the like. The microorganism or the crude enzyme product derived from the microorganism may be immobilized in advance on a resin, gel, sponge, support or the like. Immobilization is preferred because it facilitates the separation and reuse of microorganisms and crude enzyme components from the sugar solution. In particular, in the case of immobilization of microorganisms, a cellulose sponge having excellent adhesion of microorganisms without assimilation of glucose and / or xylose is preferable.

The sugar solution obtained in the present invention is preferably subjected to a membrane concentration and / or evaporation concentration step to increase the sugar concentration. Increasing the sugar concentration is preferable because it can be preferably used for the production of chemicals using the obtained sugar solution as a raw material, and leads to stability in storage and reduction in transportation costs.

Membrane concentration is preferably concentration using a nanofiltration membrane and / or a reverse osmosis membrane. Further, as a preferred example of membrane concentration, a concentrated sugar in which a sugar component is concentrated as a non-permeate by filtering through a nanofiltration membrane and / or a reverse osmosis membrane, which is a method described in WO2010 / 067785. A liquid can be obtained.

The nanofiltration membrane is also called a nanofilter (nanofiltration membrane, NF membrane), and is a membrane generally defined as “a membrane that transmits monovalent ions and blocks divalent ions”. . It is a membrane that is considered to have a minute gap of about several nanometers, and is mainly used to block minute particles, molecules, ions, salts, and the like in water.

The reverse osmosis membrane is also called an RO membrane, and is a membrane generally defined as “a membrane having a desalting function including monovalent ions”. It is a membrane that is thought to have ultrafine pores of several angstroms to several nanometers, and is mainly used for removing ionic components such as seawater desalination and ultrapure water production.

As a material of the nanofiltration membrane and / or reverse osmosis membrane used in the present invention, a composite membrane (hereinafter also referred to as cellulose acetate-based reverse osmosis membrane) or a polyamide functional layer made of a cellulose acetate-based polymer is used. And a composite membrane (hereinafter also referred to as a polyamide-based reverse osmosis membrane). Here, as the cellulose acetate-based polymer, organic acid esters of cellulose such as cellulose acetate, cellulose diacetate, cellulose triacetate, cellulose propionate, cellulose butyrate and the like, or a mixture thereof and those using mixed esters can be mentioned. It is done. The polyamide includes a linear polymer or a crosslinked polymer having an aliphatic and / or aromatic diamine as a monomer. Moreover, it is not limited to the film | membrane comprised by the said 1 type of raw material, The film | membrane containing a some film | membrane raw material may be sufficient.

The spiral membrane element is preferably used for the nanofiltration membrane. Specific examples of preferable nanofiltration membrane elements include, for example, GE Osmonics GEsepa, which is a cellulose acetate nanofiltration membrane element, Alfa Laval nanofiltration membrane element NF99 or NF99HF having a functional layer of polyamide, and crosslinked piperazine Nanofiltration membrane element manufactured by Filmtec with a functional layer of polyamide NF-45, NF-90, NF-200, NF-270 or NF-400, or nanofiltration manufactured by Toray Industries, Inc., which is mainly composed of crosslinked piperazine polyamide The company's nanofiltration membrane element SU-210, SU-220, SU-600 or SU-610, including the membrane UTC60, may be mentioned, more preferably NF99 or NF99HF, NF-45, NF-90, NF-200 or NF -400, yes SU-210, SU-220, a SU-600 or SU-610, more preferably from SU-210, SU-220, SU-600 or SU-610.

Specific examples of the reverse osmosis membrane include, for example, ultra-low pressure type SUL-G10, SUL-G20, low pressure type SU-710, SU-720, SU-720F, which are polyamide-based reverse osmosis membrane modules manufactured by Toray Industries, Inc. In addition to SU-710L, SU-720L, SU-720LF, SU-720R, SU-710P, SU-720P, high pressure types including UTC80 as a reverse osmosis membrane, SU-810, SU-820, SU-820L, SU- 820FA, Cellulose acetate reverse osmosis membrane SC-L100R, SC-L200R, SC-1100, SC-1200, SC-2100, SC-2200, SC-3100, SC-3200, SC-8100, SC-8200, Nitto NTR-759HR, NTR-729HF, NTR-70SWC, E manufactured by Denko Co., Ltd. 10-D, ES20-D, ES20-U, ES15-D, ES15-U, LF10-D, Alfa Laval RO98pHt, RO99, HR98PP, CE4040C-30D, GE GE Sepa, Filmtec BW30-4040, TW30- 4040, XLE-4040, LP-4040, LE-4040, SW30-4040, SW30HRLE-4040, KOCH TFC-HR, TFC-ULP, TRISEP ACM-1, ACM-2, and ACM-4.

The sugar solution obtained in the present invention has an advantage that a nanofiltration membrane and / or a reverse osmosis membrane is easy compared to a conventional sugar solution that does not undergo a fermentation inhibitor decomposition treatment. This is presumed to be related to a decrease in fermentation inhibiting substances in the sugar solution, but the detailed factors are unknown.

Evaporation concentration is a technique for increasing the concentration of a sugar solution by heating and / or depressurizing the sugar solution to gasify and remove the water in the sugar solution. Examples of common devices include an evaporator, a flash evaporator, a multi-effect can, spray drying, freeze drying, and the like.

The sugar solution obtained in the present invention contains monosaccharides or polysaccharides contained in an aqueous sugar solution, and includes glucose, xylose, arabinose, mannose, sucrose, cellobiose, lactose, xylobiose, xylotriose, and the like. . As a method for analyzing the sugar component in these sugar solutions, it can be quantified by HPLC and comparison with a standard product.

The sugar liquid of the present invention may be made into a sugar solid by membrane concentration and / or evaporation concentration. The saccharide solid refers to a solid that has a water content of less than 10%, preferably less than 5%, by removing water from the sugar liquid of the present invention. By using a sugar solid, the water or water activity in the sugar is reduced, and there is an advantage that contamination by microorganisms can be reduced, and further, the cost of transporting sugar can be reduced. In addition, the sugar solid obtained from the sugar liquid obtained by the method for producing a sugar liquid of the present invention is characterized by low hygroscopicity. Due to its low hygroscopicity, it has a practical advantage that it can be used stably as an industrial raw material because there is little change in quality during storage and transportation.

The sugar liquid or sugar solid of the present invention contains one or more free amino acids selected from the group consisting of serine, threonine, asparagine, aspartic acid, glutamine, glutamic acid, proline, phenylalanine, lysine and histidine which are impurities. Is below the detection limit. This is because the free amino acids contained in a small amount in the aqueous sugar solution are decomposed by the microorganisms used in the present invention that do not assimilate glucose and / or xylose or the result of degradation of the fermentation inhibitor by the crude enzyme product derived from the microorganisms. It is thought that it was used for the growth and / or metabolism of On the other hand, an amino acid contained in a peptide or polypeptide state has a characteristic that a certain amount remains in a sugar solution or a sugar solid. Therefore, when the microorganisms are grown using the sugar solution or sugar solid of the present invention as a fermentation raw material, amino acids in the peptide or polypeptide state can be used as a nutrient source for microbial growth.

Quantification of free amino acids contained in the sugar liquid or sugar solid of the present invention is preferably measured using an amino acid analyzer commercially available by the ninhydrin method. For free amino acid analysis, after adding 250 μL of 2% sulfosalicylic acid to about 2 mg of saccharide solid or saccharide solid obtained by drying the saccharide solution, stirring, sonicating for 10 minutes, adjusting the measurement sample solution, Separation and quantification can be performed with an amino acid analyzer using 25 μL of the solution. The amino acid analyzer is preferably manufactured by Hitachi, Ltd., and the amino acid analyzer L-8800A is most preferable.

Particularly for sugar solids, the content of one or more free amino acids selected from the group consisting of serine, threonine, asparagine, aspartic acid, glutamine, glutamic acid, proline, phenylalanine, lysine and histidine is below the detection limit. Thus, preferably all these free amino acids are below the detection limit, resulting in a feature that the saccharide solids are significantly less hygroscopic.

The sugar solution or sugar solid obtained by the method for producing a sugar solution of the present invention can be produced by culturing microorganisms using these as fermentation raw materials and converting the sugar into a chemical product. Specific examples of chemical products include substances that are mass-produced in the fermentation industry, such as alcohols, organic acids, amino acids, and nucleic acids. For example, ethanol, alcohol such as 1,3-propanediol, 1,4-butanediol, glycerol, acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, itaconic acid, citric acid and other organic acids, inosine, guanosine, etc. Nucleosides, nucleotides such as inosinic acid and guanylic acid, and amine compounds such as cadaverine. Furthermore, it can be applied to the production of enzymes, antibiotics, recombinant proteins, and the like.

When the sugar solution obtained in the present invention is used as a fermentation raw material for the production of chemical products, it contains a nitrogen source, inorganic salts, and if necessary, organic micronutrients such as amino acids and vitamins as necessary. Also good. In some cases, in addition to xylose, as a carbon source, sugars such as glucose, sucrose, fructose, galactose, and lactose, starch saccharified solution containing these sugars, sweet potato molasses, sugar beet molasses, high test molasses, or acetic acid An acid or an alcohol such as ethanol, glycerin or the like may be added and used as a fermentation raw material. Nitrogen sources include ammonia gas, aqueous ammonia, ammonium salts, urea, nitrates, and other supplementary organic nitrogen sources such as oil cakes, soybean hydrolysates, casein degradation products, other amino acids, vitamins, Corn steep liquor, yeast or yeast extract, meat extract, peptides such as peptone, various fermented cells and hydrolysates thereof are used. As inorganic salts, phosphates, magnesium salts, calcium salts, iron salts, manganese salts, and the like can be appropriately added.

As a method for culturing microorganisms, known fermentation culture methods such as batch culture, fed-batch culture, and continuous culture can be used. In particular, the sugar solution and / or concentrated sugar solution of the present invention has a feature that solids are completely removed by an ultrafiltration membrane or the like, and microorganisms used for fermentation can be subjected to centrifugation, membrane separation, etc. It can be separated and recovered by the method and reused. Such separation and recovery of microorganisms may be performed by continuously separating and recovering microorganisms while adding a new sugar solution and / or concentrated sugar solution during the culture period. It may be reused for the next batch culture.

Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these.

(Reference Example 1) Measurement of sugar concentration Glucose and xylose concentrations contained in a sugar solution were quantified by comparison with a sample under the HPLC conditions shown below.
Column: Luna NH ₂ (Phenomenex)
Mobile phase: Milli Q: Acetonitrile = 25: 75 (flow rate 0.6 mL / min)
Reaction solution: None Detection method: RI (differential refractive index)
Temperature: 30 ° C.

(Reference Example 2) Analysis of Fermentation Inhibitory Substance Aromatic compounds and furan compounds contained in the hydrolyzate were quantified by comparison with a sample under the HPLC conditions shown below. Each analysis sample was centrifuged at 3500 G for 10 minutes, and the supernatant component was subjected to the following analysis.
Column: Synergi HideRP 4.6 mm × 250 mm (Phenomenex)
Mobile phase: Acetonitrile-0.1% H ₃ PO ₄ (flow rate 1.0 mL / min)
Detection method: UV (283 nm)
Temperature: 40 ° C.

Acetic acid and formic acid were quantified by comparison with a standard under the following HPLC conditions. Each analysis sample was centrifuged at 3500 G for 10 minutes, and the supernatant component was subjected to the following analysis.
Column: Shim-Pack and Shim-Pack SCR101H (manufactured by Shimadzu Corporation) mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
Detection method: electric conductivity temperature: 45 ° C.

(Reference Example 3) Preparation of Trichoderma-derived cellulase Trichoderma-derived cellulase was prepared by the following method.

[Pre-culture]
Corn steep liquor 5% (w / vol), glucose 2% (w / vol), ammonium tartrate 0.37% (w / vol), ammonium sulfate 0.14 (w / vol), potassium dihydrogen phosphate 0.2 % (W / vol), calcium chloride dihydrate 0.03% (w / vol), magnesium sulfate heptahydrate 0.03% (w / vol), zinc chloride 0.02% (w / vol) , Iron (III) chloride hexahydrate 0.01% (w / vol), copper (II) sulfate pentahydrate 0.004% (w / vol), manganese chloride tetrahydrate 0.0008% ( w / vol), boric acid 0.0006% (w / vol), hexamolybdenum hexamolybdate tetrahydrate 0.0026% (w / vol) and added to distilled water, 100 mL is a triangle with 500 mL baffle Stick to flask, 121 Autoclaved at 15 ° C for 15 minutes. After allowing to cool, PE-M and Tween 80, each autoclaved at 121 ° C. for 15 minutes, were added 0.01% (w / vol). The preculture medium was inoculated with Trichoderma reesei PC3-7 at 1 × 10 ⁵ cells / mL, and cultured with shaking at 28 ° C. for 72 hours at 180 rpm (shaking apparatus: TAITEC). BIO-SHAKER BR-40LF).

[Main culture]
Corn steep liquor 5% (w / vol), glucose 2% (w / vol), cellulose (Avicel) 10% (w / vol), ammonium tartrate 0.37% (w / vol), ammonium sulfate 0.14% ( w / vol), potassium dihydrogen phosphate 0.2% (w / vol), calcium chloride dihydrate 0.03% (w / vol), magnesium sulfate heptahydrate 0.03% (w / vol) ), Zinc chloride 0.02% (w / vol), iron (III) chloride hexahydrate 0.01% (w / vol), copper (II) sulfate pentahydrate 0.004% (w / vol) ), Manganese chloride tetrahydrate 0.0008% (w / vol), boric acid 0.0006% (w / vol), hexamolybdate hexaammonium tetrahydrate 0.0026% (w / vol) Added to distilled water, 2.5L is stirred for 5L The mixture was placed in a stirring jar (DPC-2A manufactured by ABLE) and autoclaved at 121 ° C. for 15 minutes. After standing to cool, 0.1% each of PE-M and Tween 80 autoclaved at 121 ° C. for 15 minutes, respectively, was added, and Trichoderma reesei PC3-7 pre-cultured in a liquid medium by the above-described method in advance was added. 250 mL was inoculated. Thereafter, the cells were cultured at 28 ° C., 87 hours, 300 rpm, and aeration volume of 1 vvm. After centrifugation, the supernatant was subjected to membrane filtration (Millipore-made Stericup-GV material: PVDF). 1/100 amount of β-glucosidase (Novozyme 188) as a protein weight ratio was added to the culture solution adjusted under the above-mentioned conditions, and this was used as a Trichoderma-derived cellulase in the following examples.

(Example 1) Preparation of cellulose-containing biomass pretreatment product Preparation of cellulosic biomass pre-treatment product 1 (ammonia treatment)
Rice straw was used as the cellulose-containing biomass. The rice straw was put into a small reactor (manufactured by pressure-resistant glass industry, TVS-N2 30 ml) and cooled with liquid nitrogen. Ammonia gas was flowed into the reactor, and the sample was completely immersed in liquid ammonia. The reactor lid was closed and left at room temperature for about 15 minutes. Subsequently, it processed in the 150 degreeC oil bath for 1 hour. After the treatment, the reactor was taken out from the oil bath, and immediately after ammonia gas leaked in the fume hood, the reactor was further evacuated to 10 Pa and dried. This was used as a biomass pretreatment product 1 in the following examples.

2. Preparation of cellulose-containing biomass pretreatment product 2 (hydrothermal treatment)
Rice straw was used as the cellulose-containing biomass. The rice straw was soaked in water and autoclaved (made by Nitto Koatsu Co., Ltd.) for 20 minutes at 210 ° C. with stirring. After the treatment, the solution component (hereinafter, hydrothermal treatment liquid) and the treated biomass component were subjected to solid-liquid separation using centrifugation (3000 G). The obtained hydrothermal treatment liquid was used as a biomass pretreatment product 2 in the following examples.

(Example 2) Hydrolysis of cellulose-containing biomass (pretreatment product of Example 1) Trichoderma prepared in Reference Example 3 by adding distilled water to the biomass pretreatment product 1 (0.5 g) prepared in Example 1 Origin cellulase (0.5 mL) was added, distilled water was further added so that the total weight became 10 g, and the pH was adjusted to 4.5 to 5.3 with diluted sulfuric acid or diluted caustic soda. In addition, 0.1 mL of Trichoderma-derived cellulase adjusted in Reference Example 3 was added to 10 g of the hydrothermal treatment solution adjusted in Example 1, and the total weight was adjusted to 10.1 g, and the pH was 4.5. It was adjusted with dilute sulfuric acid or dilute caustic soda to be in the range of ˜5.3.

The above-mentioned two kinds of compositions adjusted for pH are transferred to a branch test tube (Tokyo Rika Kikai Co., Ltd., φ30 NS14 / 23), and this composition is transferred to a branching reaction vessel (Tokyo Rika Kikai Co., Ltd., φ30 NS14 / 23). ), And hydrolyzed by stirring for 24 hours at 50 ° C. (manufactured by Tokyo Rika Kikai Co., Ltd .: small mechanical stirrer CPS-1000, conversion adapter, addition port with three-way cock, heat retaining device MG-2200). The hydrolyzate was subjected to solid-liquid separation by centrifugation (3000 G, 10 minutes), and separated into a solution component (6 mL) and a solid. The obtained solution component is further filtered through a microfiltration membrane (a syringe filter manufactured by GE), and the obtained filtrate is hydrolyzed product 1 (derived from biomass pretreated product 1) and hydrolyzed product 2 (derived from biomass pretreated product 2). ).

Also, biomass was hydrolyzed by dilute sulfuric acid treatment. Corn biomass was immersed in sulfuric acid water (0.5% by weight) as biomass, and autoclaved (manufactured by Nitto Koatsu Co., Ltd.) at 180 ° C. for 10 minutes while stirring. After the treatment, the solution component (hereinafter, dilute sulfuric acid treatment solution) and the treatment biomass component were subjected to solid-liquid separation using centrifugation (3000 G). This dilute sulfuric acid treatment liquid was further filtered through a microfiltration membrane (a syringe filter manufactured by GE), and the obtained filtrate was designated as hydrolyzate 3.

The sugar concentration (glucose and xylose concentration) and fermentation inhibitor concentration of the hydrolysates 1 to 3 were measured by the methods described in Reference Example 1 and Reference Example 2. The hydrolysis obtained from the pretreated biomass 1 was used as the hydrolyzate 1 and the hydrolyzate obtained from the hydrothermal treatment liquid was used as the hydrolyzate 2 in the following examples. Table 1 summarizes the results of sugar analysis and aromatic compound analysis of hydrolysates 1 to 3.

In the analysis of the aromatic compounds of hydrolysates 1 to 3, although any hydrolyzate contains different components or concentrations, coumaric acid, coumarinamide, ferulic acid, ferulamide, vanillin, vanillic acid, acetovanillone It was confirmed that the composition contained one or more fermentation inhibitors selected from the group consisting of furfural and 3-hydroxymethylfurfural.

(Example 3) Degradation of fermentation inhibitor by microorganisms without assimilation of glucose and / or xylose Test Bacteria and Preculture As microorganisms having no assimilation of glucose and / or xylose, microorganisms of the genus Delftia (Delfia acidovorans NBRC14950, Delftia tsuruhatensis NBRC16741) were used. Each of the test bacteria was cultured in a TB medium (pH 7), and cultured for 24 hours with shaking. After culturing, the cells were collected by centrifugation.

2. Fermentation inhibitor decomposition treatment
The hydrolyzate 1, hydrolyzate 2 and hydrolyzate 3 obtained in Example 2 were adjusted to pH 7, and the pre-cultured cells were treated with O.D. D. _It added so that it might be set to ₆₀₀ = 10. After incubating at 30 ° C. for 24 hours, the obtained sugar solution was centrifuged (15,000 rpm, 10 min), and the sugar concentration and fermentation inhibitor concentration in the obtained sugar solution supernatant were measured. The measurement results are shown in Table 2 and Table 3. Examples of the sugar solutions obtained by decomposing hydrolyzate 1, hydrolyzate 2, and hydrolyzate 3 with Delphthia acidborans are described below as “sugar solution 1DA”, “sugar solution 2DA”, and “sugar solution 3DA”. Used for. Also, the sugar solutions obtained by decomposing hydrolyzate 1, hydrolyzate 2, and hydrolyzate 3 with Delphthia turhatensis are referred to as “sugar solution 1DT”, “sugar solution 2DT”, and “sugar solution 3DT”.

As shown in Tables 2 and 3, it was confirmed that the amount of furfural, hydroxymethylfurfural, vanillin, coumaric acid, coumarinamide, ferulamide, ferulic acid, which are fermentation inhibitors, was reduced by Delphthia microorganisms. On the other hand, it was confirmed that glucose and xylose in the sugar solution did not decrease.

(Comparative example 1) Comparison by glucose and xylose-assimilating microorganisms E. coli JM109 strain (Takara Bio Inc.) and wine yeast OC2 strain were used as glucose and xylose-assimilating microorganisms. Each of the test bacteria was cultured in LB medium (pH 7), and cultured with shaking for 24 hours. After culturing for 24 hours, the cells were collected by centrifugation.

The cells cultured in the hydrolyzate 1, hydrolyzate 2 and hydrolyzate 3 obtained in Example 2 and Example 3 were subjected to O. D. _It added so that it might be set to ₆₀₀ = 10. After incubating at 30 ° C. for 24 hours, the obtained sugar solution was centrifuged (15,000 rpm, 10 min), and the sugar concentration and fermentation inhibitor concentration in the supernatant were measured. The measurement results are shown in Tables 4 and 5.

It was confirmed that most of the aromatic compounds did not decrease when E. coli was used. In wine yeast, it was confirmed that furfural and hydroxymethylfurfural were slightly reduced, but coumarinamide, coumaric acid, ferulic acid, ferulamide and vanillin were hardly reduced. It was also confirmed that glucose and xylose contained in the hydrolysates 1 to 3 were significantly reduced.

(Example 4) Ethanol fermentation test Using the sugar solution obtained in Example 3, an ethanol fermentation evaluation, which is one type of chemical product, was carried out. E. coli KO11 strain (ATCC55124 strain) was precultured in a test tube at 30 ° C. for 24 hours in LB medium (2 mL). For sugar solution 1DA, sugar solution 2DA, sugar solution 3DA, sugar solution 1DT, sugar solution 2DT, and sugar solution 3DT, yeast extract (5 g / L), peptone (10 g / L), sodium chloride 5 g / L, pH 7. The fermentation medium (2 mL) was adjusted to 0. To each fermentation medium, 100 μL of the precultured preculture was added and cultured at 30 ° C. for 24 hours. After culturing for 24 hours, the ethanol accumulation concentration produced in each sugar solution was measured by gas chromatography (Shimadzu GC-2010 capillary GC TC-1 (GL science) 15 meter L. * 0.53 mm ID, df1.5 μm Was measured and evaluated with a flame ionization detector.

(Comparative Example 2) Ethanol Fermentation Test Using the hydrolysates 1 to 3 (Example 2), ethanol fermentation evaluation, which is one type of chemical product, was performed.

Comparing the results of ethanol fermentation of Example 4 (Table 6) and the results of ethanol fermentation of Comparative Example 2 (Table 7), the ethanol fermentation of Example 4 was more reduced due to the reduction of fermentation inhibitors in step (2). The ethanol production was significantly higher.

(Example 5) Metabolic degradation of ferulamide and ferulic acid of Delphtia microorganisms In order to confirm whether degradation of aromatic compounds in the hydrolyzate of Example 3 is due to degradation by Delphtia microorganisms, ferulamide In the model reaction system to which was added, the degradation products were identified and the amount of change was traced.

Delftia acidvorans NBRC14950 and Delftia tsuruhatensis NBRC16741 were cultured in TB medium (pH 7) and invasive culture was performed for 64 hours. After culturing, each bacterial cell was collected by centrifugation. A solution obtained by dissolving ferramide in N, N-dimethylformamide was diluted with 10 mM Tris-HCl buffer to prepare a 1 g / L ferramide solution, and the pH was adjusted to 10 with sodium hydroxide. Each pre-collected microbial cell is O.D. D. _It added so that it might become ₆₀₀ = 10, and it heat-retained at 30 degreeC for 72 hours, The reaction liquid was collect | recovered with time. The obtained reaction liquid was centrifuged (15,000 rpm, 10 min), and the aromatic compound concentration was measured by the method of Reference Example 1 in the obtained reaction liquid supernatant. FIG. 3 shows the results of Delftia tsurhatensis, and FIG. 4 shows the results of Delftia acidborans. As shown in FIG. 3 and FIG. 4, in both Delphthia genera, the ferulamide concentration decreased rapidly with the start of heat retention, and the amount of ferulic acid produced reached its peak after 6 hours. Further, it was confirmed that ferulic acid disappeared within 36 hours and was converted to vanillic acid. Furthermore, it was confirmed that vanillic acid further decreased with the incubation time. From the above results, it was confirmed that in microorganisms belonging to the genus Delphia, they were sequentially converted to ferulamide, ferulic acid, and vanillic acid, and finally vanillic acid was also decomposed.

(Example 6) Metabolic degradation of coumarinamide and coumaric acid of Delphthia spp. In order to confirm whether or not the degradation of aromatic compounds in the hydrolyzate of Example 3 is due to degradation by Delphtia spp. In the model reaction system, the degradation products were identified and the amount of production was tracked.

In the same procedure as in Example 5, ferulamide was changed to coumarinamide and the metabolic pathway was identified. FIG. 5 shows the results of Delftia tsurhatensis, and FIG. 6 shows the results of Delftia acidborans. As shown in FIG. 5 and FIG. 6, it was found that in any Delphthia genus, the concentration of coumarinamide gradually decreased over 48 hours from the start of the incubation. Further, the coumaric acid concentration increased with the decrease of coumarinamide, and reached a peak at 24 hours of heat retention. In addition, as shown in FIG. 5, in Delftia thulhatensis, it was confirmed that p-hydroxybenzoic acid was produced with a decrease in coumaric acid. Similarly, the production of p-hydroxybenzoic acid was confirmed in Delphthia acid boranes in a trace amount. From the above results, it was confirmed that in microorganisms belonging to the genus Delphia, coumarinamide, coumaric acid, and p-hydroxybenzoic acid were converted in this order, and finally p-hydroxybenzoic acid was also decomposed.

(Example 7) Effect of pH in degradation of fermentation inhibitor by microorganisms belonging to the genus Delphtia The hydrolysis inhibitor 1 of Example 2 was adjusted to pH 4, pH 8.5, pH 10, and pH 12, and the fermentation inhibitor was subjected to the same procedure as in Example 4. A decomposition treatment was performed. The test bacterium was performed using Delftia tsuruhatensis. The results are shown in Table 8.

At pH 5 and pH 12, it was confirmed that there was little decrease in aromatic compounds. On the other hand, at pH 8 and pH 10, it was confirmed that the amount of the aromatic compound was decreased compared to pH 7 (Example 3), and in particular, a marked decrease was observed in coumarinamide and ferulamide. As shown in Reference Example 5, it has been confirmed that Delphthia microorganisms do not grow at pH 10, and at pH 10, Delphthia microorganisms do not grow, but the aromatic converting enzyme possessed by Delphthia microorganisms functions, and fragrance It was presumed that the decomposition of group compounds progressed.

(Reference Example 4) Optimum growth temperature of Delphtia microorganisms Optimum growth temperature of Delphtia was examined. The Delphtia tsuruhatensis of Example 3 was used as a test bacterium, and the bacterium was precultured in a test tube in an LB medium (5 mL) at 30 ° C. and 180 rpm for 24 hours. Thereafter, 50 μL of the precultured preculture was added to TB medium (5 mL, pH 7), and cultured at 30 ° C. to 50 ° C., 180 rpm, for 24 hours. After culturing for 24 hours, OD ₆₀₀ was measured. The results are shown in Table 9.

It was confirmed that the cells proliferated in the temperature range of 20 ° C to 35 ° C. However, it was confirmed that the cells did not grow above 40 ° C.

(Reference Example 5) Optimum growth pH of Delphtia microorganism
The optimum pH for growth of Delphtia microorganisms was examined. Delphthia tsuruhatensis was used as a test bacterium, and this bacterium was precultured in a test tube in an LB medium (5 mL) at 30 ° C. and 180 rpm for 24 hours. Thereafter, 50 μL of the precultured preculture was added to TB medium (5 mL, pH 7) and TB medium adjusted to pH 4.0, 8.5, and 10 with hydrochloric acid and sodium hydroxide, respectively. Incubation was carried out at 180 rpm for 24 hours. After culturing for 24 hours, OD ₆₀₀ was measured. The results are shown in Table 10.

It was confirmed that Delphthia spp. Grew up to pH 8.5 but did not grow at pH 10 at all. Similarly, no growth was observed at pH 4.

(Example 8) Step of membrane concentration (reverse osmosis membrane) of sugar solution 1300 mL each of sugar solution 1DA, sugar solution 2DA, and sugar solution 3DA described in Example 3, each having a pore size of 0.22 μm ( The resulting fine particles were removed by subjecting to Stericup-GV (Millipore). Using the reverse osmosis membrane (UTC-80, manufactured by Toray Industries, Inc.), the obtained liquid was measured with a flat membrane small crossflow filtration unit (SEPA CF-II, manufactured by GE Osmonix, effective membrane area 140 cm 2). Filtered at 0 ° C. Filtration was carried out by cross-flow filtration, adjusting as needed so that the transmembrane pressure difference was always 4 MPa, and the liquid volume was concentrated to about 1/4 (4-fold concentration). Table 8 shows the results of measuring the time required for concentration, the filtration rate at the end of concentration, and the sugar concentration after concentration. In addition, as a comparative example (Comparative Example 3), the hydrolyzate 1 to 3 described in Example 3 was used (the step of decomposing the fermentation inhibitor by a microorganism having no assimilation ability of glucose and xylose was not performed) Table 11 also shows the results of membrane concentration by the same method as described above.

As shown in Table 11, the time required for concentration of the sugar solution by 4 times was shortened in the case of the sugar solution subjected to the step of degrading the fermentation inhibitor by microorganisms having no assimilation ability of glucose and xylose. Further, the filtration rate at the end of concentration was faster when the step of degrading the fermentation inhibitor by a microorganism having no assimilation ability of glucose and xylose was performed. From this, it was found that the filterability at the time of membrane concentration of the sugar solution was improved by subjecting each hydrolyzate to the step of degrading the fermentation inhibitor by a microorganism having no ability to assimilate glucose and xylose.

(Example 9) Preparation of sugar solid 15 mL each of the sugar solution 1DA after membrane concentration described in Example 8 and the hydrolyzate 1 after membrane concentration described in Example 8 were also taken for comparison. After being transferred to a round bottom glass flask, it was frozen at −70 ° C. The frozen sugar solution and hydrolyzate 1 were lyophilized with a freeze dryer (EYLA: Tokyo Science Machine Co., Ltd.) at −45 ° C. for 48 hours. The weight after drying was 1.41 g of sugar solution 1DA after membrane concentration and 1.37 g of hydrolyzate 1.

0.5 g of the obtained saccharide solid (sugar solid 1DA: lyophilized sugar liquid 1DA after membrane concentration, saccharide solid hydrolyzate 1 (lyophilized hydrolyzate 1 after membrane concentration) in a glass vial Each bottle was collected in a bottle (13.5 mL capacity) and then left at room temperature (approximately 25 ° C.) for 24 hours with the vial lid open, and the change in appearance was compared, and the results are shown in FIG. In the solid hydrolyzate 1, moisture absorption started in the process of separating the freeze-dried product from the round bottom flask into the vial, and in the photograph immediately after opening, the color tone becomes dark and is in a lump state (right side of FIG. 7A). On the other hand, the sugar solid 1DA was still in powder form (left side of Fig. 7A), and the photograph after 24 hours after opening the lid of the vial is shown in Fig. 7B. Absorbs moisture and is no longer powdery, but sticky molasses On the other hand, in the sugar solid 1DA, a slight decrease in the apparent bulkiness was observed after 24 hours, and it was assumed that moisture absorption occurred, but the powder state was maintained. That is, the sugar solid of the present invention has a low hygroscopic property, and thus has a very high form stability under air release, and has a practical advantage. I was able to confirm.

(Example 10) Amino acid analysis The amino acid analysis contained in the sugar solid hydrolyzate 1 and sugar solid 1DA obtained in Example 9 was performed according to the following procedure. In the amino acid analysis, the free amino acid concentration was measured. The analyzer used was an amino acid analyzer L-8800A (Hitachi, Ltd.), the measurement conditions were ninhydrin method, detection wavelength: 440 nm (proline, hydroxyproline), 570 nm (amino acids other than proline, hydroxyproline) ).

From the freeze-dried product, saccharide solid hydrolyzate 1 (1.41 g) and saccharide solid 1DA (1.37 g) obtained in Example 9, 2.05 mg each was collected in a tube and 250 μL of 2% sulfosalicylic acid was added. -After stirring, sonication was performed for 10 minutes. This solution was filtered with a 0.22 μm filter to obtain a measurement sample solution. An analysis was performed under the above apparatus conditions using 25 μL of this sample solution. The analysis values are shown in Table 12.

The detected concentration is the concentration (mg) of each free amino acid contained per gram of the saccharide solid weight analyzed. The concentration conversion in the concentrated sugar solution is a value converted into a concentration present in 15 mL of each membrane concentrated sugar solution subjected to freeze-drying. In the sugar solid hydrolyzate 1, it was confirmed from analytical values that all amino acids were contained. On the other hand, in sugar solid 1DA which decomposed the fermentation inhibitor by microorganisms that do not assimilate glucose and xylose, serine, threonine, asparagine, aspartic acid, glutamine, glutamic acid, proline, phenylalanine, lysine, histidine are released. Amino acids could not be detected (not detected). Therefore, it was confirmed that the free amino acid concentration in the concentrated sugar solution before lyophilization was 0 mg / L. That is, it was confirmed that the sugar solution and sugar solid of the present invention had the above-mentioned free amino acid concentration below the detection limit.

Next, the same analysis was performed on the total amount of amino acids contained in each sugar solid. The total amount of amino acids includes free amino acids as well as amino acids that exist in the form of peptides or polypeptides. Therefore, 2.05 mg each of lyophilized product, sugar solid hydrolyzate 1 (1.41 g) and sugar solid 1DA (1.37 g) obtained in Example 9 was collected in a tube and 6 mol / L hydrochloric acid 250 μL. Was added, and after nitrogen substitution and vacuum sealing, hydrolysis was performed at 110 ° C. for 22 hours. 0.02 mol / L hydrochloric acid (200 μL) was added to the residue obtained by drying under reduced pressure and dissolved. This solution was filtered with a 0.22 μm centrifugal filtration unit to obtain a measurement sample solution. An analysis was performed under the above-described apparatus conditions using 25 μL of this sample solution. The analysis results are shown in Table 13.

It was confirmed from the analytical values that all of the amino acids are contained in any of the sugar solid hydrolyzate 1 and sugar solid 1DA in which the fermentation inhibitor was decomposed by microorganisms having no assimilation ability of glucose and xylose. . However, it was confirmed that the sugar solid 1DA was less. That is, free amino acids are mainly consumed by microorganisms that do not assimilate glucose and xylose, and amino acids present in the form of peptides or polypeptides remain unconsumed in the sugar solution. It was confirmed that The inclusion of amino acids present in such peptides or polypeptides is important in microbial fermentation production and is used as a nitrogen source for microbial growth.

(Example 11) Degradation of fermentation inhibitor by microorganisms without assimilation of glucose and xylose 2: Treatment of molasses (molasses) As molasses solution containing fermentation inhibitor, molasses (molasses: Molasses-Agri, Organic Land) Co., Ltd.). The used molasses is a mixture using about 90% sugarcane-derived molasses and about 10% sweet potato molasses as biomass. This waste molasses was diluted 6 times with RO water and autoclaved at 121 ° C. for 20 minutes. After sterilization, sodium hydroxide was added to adjust the pH to 6.7. Used as an aqueous sugar solution containing a fermentation inhibitor (waste molasses aqueous solution). Table 14 shows the analysis results of the aromatic compounds. It was confirmed that the waste molasses aqueous solution contained glucose, fructose, and sucrose as sugars and 3-hydroxymethylfurfural as a fermentation inhibitor. The sugar aqueous solution was treated with Delphthia thulhatensis according to the description in Example 3. The obtained molasses is used as molasses DT, and the analysis values (sugar and aromatic compound) are shown in Table 14. It was confirmed that 3-hydroxymethylfurfural contained as a fermentation inhibitor was completely decomposed and disappeared from the molasses DT.

The method for producing a sugar solution according to the present invention can be used for producing a sugar solution with reduced fermentation inhibition from biomass. The sugar liquid or sugar solid produced in the present invention can be used as a fermentation raw material for various chemicals.

Claims

A method for producing a sugar solution from biomass, which is a fermentation inhibitor contained in an aqueous sugar solution obtained from biomass, such as coumaric acid, coumaric acid, ferulic acid, ferulamide, vanillin, vanillic acid, acetovanillone, furfural and 3-hydroxymethyl A method for producing a sugar solution, comprising the step of decomposing one or more compounds selected from the group consisting of furfural with a microorganism that does not assimilate glucose and / or xylose or a crude enzyme derived from the microorganism.
The method for producing a sugar liquid according to claim 1, wherein the microorganism having no assimilation property of glucose and / or xylose is a Delphia sp.
The non-assimilable microorganism of glucose and / or xylose is composed of Delftia acidvorans, Delftia lacustris, Delftia tsuruhatensis and Delphia litia litia litia. The manufacturing method of the sugar liquid of Claim 1 or 2 which is 1 type, or 2 or more types chosen.
The method for producing a sugar liquid according to any one of claims 1 to 3, wherein the sugar aqueous solution is a sugar aqueous solution obtained by hydrolyzing cellulose-containing biomass.
The method for producing a sugar solution according to claim 4, comprising a step of preparing an aqueous sugar solution by one or more treatments selected from the group consisting of acid treatment, alkali treatment, hydrothermal treatment and enzyme treatment of cellulose-containing biomass.
The method for producing a sugar liquid according to any one of claims 1 to 3, wherein the aqueous sugar solution is molasses.
The method for producing a sugar liquid according to any one of claims 1 to 6, wherein the fermentation inhibiting substance decomposition treatment step is a treatment at a monosaccharide concentration of less than 100 g / L.
The method for producing a sugar solution according to any one of claims 1 to 7, wherein the fermentation inhibiting substance decomposition treatment step is treatment within a pH range of 6 to 11.
The method for producing a sugar solution according to any one of claims 1 to 8, wherein the sugar concentration obtained by the production method according to any one of claims 1 to 8 is increased by membrane concentration and / or evaporation concentration.
A method for producing a sugar solid, wherein a sugar solid is obtained by membrane concentration and / or evaporation concentration of the sugar solution obtained by the production method according to any one of claims 1 to 8.
A sugar solution obtained by the method for producing a sugar solution according to any one of claims 1 to 9.
A sugar solid obtained by the method for producing a sugar solid according to claim 10.
One or two selected from the group consisting of cellulose-containing biomass or molasses-derived molasses or sugar solids, which are impurities such as serine, threonine, asparagine, aspartic acid, glutamine, glutamic acid, proline, phenylalanine, lysine and histidine A sugar solution or a sugar solid in which the content of free amino acids of species or more is below the detection limit.
A step of obtaining a sugar solution by the method for producing a sugar solution according to any one of claims 1 to 9, and a step of culturing microorganisms and converting the sugar into a chemical product using the obtained sugar solution as a fermentation raw material, Manufacturing method of chemical products.
A method for producing a chemical product, comprising the steps of obtaining a sugar solid by the method for producing a sugar solid according to claim 10, and culturing microorganisms and converting the sugar into a chemical product using the obtained sugar solid as a fermentation raw material. .